High-dose N-acetylcysteine decreases silica-induced lung fibrosis in the rat

Abstract

Objective

To study the potential of high-dose N-acetylcysteine (NAC) to attenuate silica-induced pulmonary fibrosis in the rat.

Methods

Rats exposed to intratracheal instillation of silica particles were treated with 500 mg/kg NAC orally every day for 7 days, before and up to 28 days after silica administration (n = 32), or received no treatment following silica exposure (n = 32); a third group received intratracheal saline (n = 32). Fibrosis score, and hydroxyproline (HYP) and malondialdehyde (MDA) content, were assessed in lung tissue. Bronchoalveolar lavage fluid (BALF) and serum levels of tumour necrosis factor (TNF)-α, interleukin (IL)-8 and high-sensitivity C-reactive protein (hsCRP) were assessed by enzyme-linked immunosorbent assay.

Results

Histopathology revealed inflammation and fibrosis in lung tissue from rats exposed to silica, but not in saline controls. The fibrosis score was significantly lower in animals treated with NAC compared with silica-exposed untreated rats. HYP and MDA content were significantly lower at all timepoints, following NAC treatment versus no treatment, in silica-exposed rats. NAC attenuated silica-induced increases in TNF-α, IL-8 and hsCRP in BALF and serum.

Conclusions

Oral treatment with high-dose NAC during early silica exposure can ameliorate the activity of proinflammatory cytokines, thus attenuating subsequent lung fibrosis. These results suggest that NAC has potential as a treatment for silica-induced lung fibrosis.

Introduction

Silicosis, caused by the inhalation of fine particles of crystalline silicon dioxide (silica), is an important lung pneumoconiosis. It is a potentially fatal, irreversible, pulmonary disease that is characterized by interstitial inflammation and fibrosis, which is typified by the recruitment of phagocytic cells to the lung. ¹ Despite extensive research efforts, the exact mechanism of silicosis has not yet been fully elucidated. Silicosis is believed to be initiated by the phagocytosis of silica particles by alveolar macrophages; these macrophages release numerous oxidants and cytokines, which play key roles in the development and progression of the disease. ²

Despite the development of several therapies for silicosis (including whole-lung lavage, steroids, tetrandrine drugs and aluminum powder inhalation), ³ none can reverse or slow the disease process. Novel approaches for the prevention and treatment of silica-induced inflammation and fibrosis are needed.

The thiol compound N-acetylcysteine (NAC) is used as an antioxidant and mucolytic drug; NAC has been found to be beneficial in the management of chronic bronchitis ⁴ and acetaminophen overdose, ⁵ and in the prevention of radiocontrast-induced nephropathy. ⁵ The incidence of NAC-associated adverse effects is low. ⁶ NAC is a precursor of glutathione and directly scavenges oxygen free radicals.^7,8 It can regulate the production of some cytokines and suppress the activation of transcription factors, such as nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) to modulate cell-signalling pathways. ⁹ NAC has been used with varying success to enhance lung glutathione levels and reduce inflammation in patients with chronic obstructive pulmonary disease and interstitial pulmonary fibrosis.^10–13It is also effective in attenuating amiodarone-induced lung fibrosis ¹⁴ and radiation-induced lung injury and fibrosis. ¹⁵

The present study aimed to examine the effects and mechanism of action of orally-administered high-dose NAC in a rat model of lung inflammation and fibrosis, induced by intratracheal instillation of silica dust suspension.

Materials and methods

Animals

Male, pathogen-free Wistar rats (n = 96), aged 6–8 weeks (weight 180–220 g), were obtained from the Animal Experiment Centre, Shandong University, Jinan, China, and kept on a 12-h light/12-h dark cycle with food and water freely available. The animal experimental protocol complied with the Animal Management Rules of the Chinese Ministry of Health (document no. 55, 2001) and was approved by the Animal Care Committee, Shandong University.

Study treatment and surgical procedures

Animals were randomly divided into three treatment groups (n = 32 per group): model (intratracheal instillation of silica particles alone); treatment (intratracheal instillation of silica particles plus NAC treatment); control (intratracheal administration of saline alone).

Crystalline silica (Sigma-Aldrich, St Louis, MO, USA) particles (primary diameter 1 – 5 µm) were sterilized by heating at 160℃ for 90 min before suspension in sterile saline at 50 mg/ml. Silica suspensions (0.5 ml) were delivered into rat lungs by intratracheal instillation following tracheotomy, in animals anesthetized with 40 mg/kg pentobarbital sodium, administered via intraperitoneal (i.p.) injection. Control rats underwent the same procedure but received an equal volume of saline instead of silica.

Rats received 500 mg/ kg NAC in a final volume of 2 ml distilled water as a single oral dose every day, from 7 days before intratracheal silica instillations until the time of analysis.

Animals were sacrificed using sodium pentobarbital i.p. injection (120 mg/rat) at 3, 7, 14 or 28 days after silica instillation; blood samples were taken from the abdominal aorta at these times. Serum was separated by centrifugation at 1372  g for 10 min at 4℃. Serum samples were stored at –70℃ until use.

BALF collection

The right lung was ligated at the bronchi with surgical wire and bronchoalveolar lavage fluid (BALF) was obtained by washing the left lung three times with 2-ml aliquots of saline at 37℃ through a tracheal cannula. BALF was centrifuged at 112  g for 10 min at 4℃ and was used for enzyme-linked immunosorbent assays (ELISA). BALF samples were stored at –70℃ until use.

Histopathology of lung specimens

The right lung lobes were removed, blotted dry and weighed before the middle lobe was fixed in 4% formalin. Specimens were embedded in paraffin wax and sectioned at 4 µm prior to staining with haematoxylin and eosin (to examine histopathology) and Masson's trichrome (to examine collagen deposition). Fibrotic findings in each section were examined by light microscopy and scored according to the method of Ashcroft et al. ¹⁶ on a scale between 0 and 8 (where 0, normal lung; 8, total fibrous obliteration of the microscope field) by examining >15 successive fields at a magnification of ×200. To avoid observer bias, three experienced histopathologists, blinded to the treatment groups, interpreted the images independently; the mean of their findings was considered to be the fibrotic score of the specimen.

Collagen and lipid peroxidation assays

On removal, the other half of the right lung specimen was frozen immediately at −80℃ for determination of hydroxyproline (HYP) and malondialdehyde (MDA) levels. Lung tissue was homogenized using an ultrasonic sonochemistry homogenizer (U & Star Industry, Hangzhou, Zhejiang, China). Collagen deposition was estimated by measuring the HYP content in lung homogenates by alkaline hydrolysis analysis, using a commercially-available kit (Nanjing Jiancheng Bioengineering Inst., Nanjing City, Jiangsu, China) according to the manufacturer’s instructions. Lipid peroxidation, as determined by the formation of MDA, was measured by the thiobarbituric acid method using a commercially-available kit (Nanjing Jiancheng Bioengineering Inst.). Samples (1 ml) underwent spectrophotometry using a nucleic acid/protein analyser (DU800, Beckman Coulter, Brea, CA, USA) with absorbance at 532 nm.

ELISA to determine cytokine levels in BALF and serum

Levels of tumour necrosis factor (TNF)-α and interleukin (IL)-8 were measured in BALF and serum using commercial ELISA kits (Wuhan Boster Biological Technology, Wuhan, Hubei, China). High-sensitivity C-reactive protein (hsCRP) in BALF and serum was assayed using a highly-sensitive ELISA assay kit (Diagnostic Systems Laboratories, Chicago, IL, USA). All ELISA kits were used according to the manufacturers’ instructions.

Statistical analyses

All statistical analyses were carried out using SPSS® statistical software, version 13.0 (SPSS Inc., Chicago, IL, USA). Data were expressed as mean ± SE. Between-group differences were determined using one-way analysis of variance (ANOVA). Post hoc comparisons were carried out using the least significant difference test when equal variances were assumed, or with Dunnett’s test when equal variances were not assumed. A P-value of< 0.05 was considered statistically significant.

Results

Representative sections of rat lung tissue are shown in Figure 1. A normal alveolar structure, without cellular infiltration or fibrous thickening, was observed in control rats (Figures 1I and 1J). At day 3 after particle instillation, acute inflammation (as demonstrated by infiltration of neutrophils into the alveoli and thickening of the alveolar wall) were observed in lung tissue from the silica model group (Figure 1A). By day 7, specimens showed alveolar thickening with infiltration of macrophages, lymphocytes and neutrophils (Figure 1B). In animals treated with NAC, the severity of alveolitis was lower compared with the untreated rats on days 3 and 7 (Figures 1E and 1F). On day 14, animals in the untreated silica-exposed group showed pulmonary lesions consisting of multifocal areas of intense fibrosis (data not shown). The number of neutrophils was decreased and the number of lymphocytes was increased, compared with day 7: granulomas containing macrophages, neutrophils and fibroblasts appeared to be larger and more numerous. By day 28, the untreated silica-exposed model group showed diffuse fibrosis with destruction of the alveolar structure (Figure 1C). The extent of fibrotic lesions and inflammation on day 28 was reduced in animals treated with NAC compared with the untreated model (Figure 1G).

Figure 1.

Representative sections (4 -µm thickness) of rat lung tissue stained with haematoxylin and eosin (H&E) for histopathology, or Masson’s trichrome for collagen deposition (original magnification × 200). (A – C) H&E staining, silica model group, days 3, 7 and 28, respectively; (D) Masson’s trichrome staining, silica model group, day 14; (E – G) H&E staining, N-acetylcysteine (NAC)-treated silica model group, days 3, 7 and 28, respectively; (H) Masson’s trichrome staining, NAC-treated silica model group, day 14; (I) H&E staining, control group, day 3; (J) Masson’s trichrome staining, control group, day 14; (K) Pulmonary fibrosis as determined by the Ashcroft score ¹⁶ (where 0, normal lung; 8, total fibrous obliteration of the microscope field) on days 14 and 28 after instillation of silica or saline control. Data presented as mean ± SE (n = 8). ^*P < 0.05 versus control/ untreated silica model (one-way analysis of variance and least significant difference test). The colour version of this figure is available at: http://imr.sagepub.com.

In the untreated model group, Masson’s trichrome staining revealed weak collagen deposition at day 3; increases in collagen deposition began at day 7 following particle instillation and were even more pronounced on days 14 (Figure 1D) and 28. The degree of fibrosis was reduced in animals treated with NAC compared with the untreated model on days 7, 14 (Figure 1H) and 28.

Compared with the untreated animals exposed to silica, the Ashcroft score for fibrosis ¹⁶ was lower in those animals treated with NAC on days 14 and 28 (P < 0.05, Figure 1K).

Hydroxyproline levels increased significantly from day 3 onwards (P < 0.05) following silica exposure compared with lung homogenates from control animals. Treatment with NAC significantly reduced the HYP content at days 7, 14 and 28 (P < 0.05, Figure 2A). Increases in lung MDA peaked at day 7 following silica exposure before declining again until the end of the experiment; levels remained significantly higher compared with control animals at all timepoints (P < 0.05). MDA content was significantly reduced by NAC treatment compared with silica exposure alone, at all timepoints (P < 0.05, Figure 2B).

Figure 2.

Levels of (A) hydroxyproline (HYP) and (B) malondialdehyde (MDA) in lung homogenates, (C) tumour necrosis factor (TNF)-α in serum, and (D) interleukin (IL)-8 in bronchoalveolar lavage fluid (BALF) in rats exposed to silica (model group), exposed to silica and treated with N-acetylcysteine (NAC treatment), or exposed to saline (control group). Data presented as mean ± SE (n = 8). ^*P < 0.05 versus control/ untreated silica model (one-way analysis of variance and least significant difference test). The colour version of this figure is available at: http://imr.sagepub.com.

To ensure experimental accuracy, BALF recovery should be > 80%; In our study, BALF recovery was > 80% in all groups and was not significantly different between groups. In the untreated silica-exposure group, serum TNF-α levels began to increase from day 3 after silica instillation, peaked on day 7, and reached a plateau until the end of the experiment on day 28 (Figure 2C); BALF levels of TNF-α were high on day 3, peaked on day 14 and reduced thereafter. NAC treatment attenuated elevated TNF-α in both BALF and serum (P < 0.05,). Levels of IL-8 in BALF (Figure 2D) and serum from the untreated model group peaked on day 3 and rapidly decreased thereafter. These levels were significantly attenuated by NAC treatment (P < 0.05). Compared with control rats, levels of hsCRP were increased in BALF and serum from rats exposed to silica (data not shown). Levels in BALF and serum decreased from day 3 to day 14. Any increases in hsCRP were reduced in NAC-treated animals.

Discussion

In the present study, a rat model of silica-induced fibrosis was established by intratracheal instillation of a silica particle suspension, to determine levels of inflammatory cytokines in BALF and serum as a measure of pulmonary damage, inflammation and fibrosis.¹⁷⁻²⁰ IL-8, which is considered to be a principle mediator of inflammation, is produced by monocytes, T-lymphocytes, neutrophils, fibroblasts, and epithelial and endothelial cells. ²¹ TNF-α and IL-8 mediate the recruitment and activation of inflammatory cells, and stimulate fibroblast proliferation and collagen production.^22–24 Histopathological analyses in the present study revealed early alveolitis in the silica model, characterized by infiltration by alveolar macrophages and other leucocytes (especially neutrophils), accompanied by early thickening of the alveolar walls. Quantities of infiltrates, organized granulomas and collagen deposition increased over time indicating progressive pulmonary inflammation and damage compared with the control group. Silicosis is associated with inflammatory changes within the alveoli, ²⁵ which may result in pulmonary fibrosis. In the present study, lipid peroxidation (as measured by MDA content in lung homogenates) increased early during silica damage; in addition, fibrosis was indicated by a significant increase in HYP content. Oxidant stress and the inflammatory response following exposure to silica particles, therefore, appeared to be an initiating step in the pathogenesis of fibrosis in this study.

Findings from the present study support the following basic mechanisms involved in the aetiology of silicosis: ²⁶ oxidant production by pulmonary phagocytes causing lipid peroxidation, cell injury and lung scarring; release of mediators from alveolar macrophages and epithelial cells that facilitate recruitment of polymorphonuclear leucocytes and macrophages, leading to increases in proinflammatory cytokines and reactive species, and lung injury and scarring. Silica-induced oxidative stress can also activate specific transcription factors, including NF-κB and activator protein-1.^27,28 One outcome of transcription-factor activation can be increased cytokine expression, which in turn induces neutrophil recruitment and the further activation of NF-κB and activator protein-1, thereby augmenting the inflammatory response and tissue damage.^29,30 Further research is needed to evaluate the inflammatory mediators involved in silica-induced lung fibrosis.

N-acetylcysteine has therapeutic potential in inflammatory diseases. The effect of NAC on the fibrogenicity of silica was investigated in the present study and the pathological mechanisms underlying these effects were explored. Results from the present study showed that inflammatory activity was reduced by NAC in a rat model of silica-induced fibrosis by inhibiting the production of the inflammatory mediators TNF-α, IL-8 and hsCRP, which resulted in significant attenuation of fibrosis. It is possible that NAC may mediate these effects through suppression of transcription factors such as NF-κB or by direct scavenging of reactive oxygen radicals. The present results support the hypothesis that NAC interruption of the inflammatory cascade before irreversible tissue injury has occurred may ultimately prevent fibrosis.

In conclusion, the present study demonstrated that NAC administration inhibited pulmonary inflammation and fibrosis induced by silica in a rat model. Furthermore, NAC significantly reduced the levels of inflammatory cytokines in BALF and serum in animals exposed to silica and may, therefore, represent a potential therapy for silicosis.